Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

BACKGROUND: Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. METHODS: Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. RESULTS: Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. CONCLUSIONS: sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

Impact of microglia isolation and culture methodology on transcriptional profile and function.

BACKGROUND: Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate five different microglia isolation methods-three variants of microglia isolation from neonatal mice and two variants of microglia isolation from adult mice-on transcriptional profile and response to HMGB1. METHODS: Microglia from neonatal mice, age 0-3 days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al. in Front Cell Neurosci 17:1082180, 2023), or acute isolation using CD11b beads (Bordt et al. in STAR Protoc 1:100035, 2020. https://doi.org/10.1016/j.xpro.2020.100035 ). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1ß, and IFNß concentration in supernatant by ELISA and assessing gene expression patterns using bulk mRNA sequencing. RESULTS: All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription and secretion of TNFα, IL1ß, and IFNß in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from neonates than from adult mice, with sparse changes among the three MGC culturing conditions. Additionally, cultured microglia derived from adult and microglia derived from MGCs from neonates display transcriptional signatures corresponding to an earlier developmental stage. CONCLUSION: These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response.

Acetylcholine is produced in the spleen in response to vagus nerve activation; however, the effects on antibody production have been largely unexplored. Here, we use a chronic vagus nerve stimulation (VNS) mouse model to study the effect of VNS on T-dependent B cell responses. We observed lower titers of high-affinity IgG and fewer antigen-specific germinal center (GC) B cells. GC B cells from chronic VNS mice exhibited altered mRNA and protein expression suggesting increased apoptosis and impaired plasma cell differentiation. Follicular dendritic cell (FDC) cluster dispersal and altered gene expression suggested poor function. The absence of acetylcholine-producing CD4+ T cells diminished these alterations. In vitro studies revealed that α7 and α9 nicotinic acetylcholine receptors (nAChRs) directly regulated B cell production of TNF, a cytokine crucial to FDC clustering. α4 nAChR inhibited coligation of CD19 to the B cell receptor, presumably decreasing B cell survival. Thus, VNS-induced GC impairment can be attributed to distinct effects of nAChRs on B cells.

Extrarenal symptoms associate with worse quality of life in patients enrolled in the AMP RA/SLE Lupus Nephritis Network.

OBJECTIVE: Lupus nephritis (LN) can occur as an isolated component of disease activity or be accompanied by diverse extrarenal manifestations. Whether isolated renal disease is sufficient to decrease health related quality of life (HRQOL) remains unknown. This study compared Patient-Reported Outcomes Measurement Information System 29-Item (PROMIS-29) scores in LN patients with isolated renal disease to those with extrarenal symptoms to evaluate the burden of LN on HRQOL and inform future LN clinical trials incorporating HRQOL outcomes. METHODS: A total of 181 LN patients consecutively enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership completed PROMIS-29 questionnaires at the time of a clinically indicated renal biopsy. Raw PROMIS-29 scores were converted to standardized T scores. RESULTS: Seventy-five (41%) patients had extrarenal disease (mean age 34, 85% female) and 106 (59%) had isolated renal (mean age 36, 82% female). Rash (45%), arthritis (40%) and alopecia (40%) were the most common extrarenal manifestations. Compared with isolated renal, patients with extrarenal disease reported significantly worse pain interference, ability to participate in social roles, physical function, and fatigue. Patients with extrarenal disease had PROMIS-29 scores that significantly differed from the general population by > 0.5 SD of the reference mean in pain interference, physical function, and fatigue. Arthritis was most strongly associated with worse scores in these three domains. CONCLUSION: Most patients had isolated renal disease and extrarenal manifestations associated with worse HRQOL. These data highlight the importance of comprehensive disease management strategies that address both renal and extrarenal manifestations to improve overall patient outcomes.

Immune cell expression patterns of CD39/CD73 ectonucleotidases in rodent models of cardiac arrest and resuscitation.

Background: Cardiac arrest (CA) is a significant public health concern. There is the high imminent mortality and survival in those who are resuscitated is substantively compromised by the post-CA syndrome (PCAS), characterized by multiorgan ischemia-reperfusion injury (IRI). The inflammatory response in PCAS is complex and involves various immune cell types, including lymphocytes and myeloid cells that have been shown to exacerbate organ IRI, such as myocardial infarction. Purinergic signaling, as regulated by CD39 and CD73, has emerged as centrally important in the context of organ-specific IRI. Hence, comprehensive understanding of such purinergic responses may be likewise imperative for improving outcomes in PCAS. Methods: We have investigated alterations of immune cell populations after CA by utilizing rodent models of PCAS. Blood and spleen were collected after CA and resuscitation and underwent flow cytometry analysis to evaluate shifts in CD3+CD4+ helper T cells, CD3+CD8a+ cytotoxic T cells, and CD4/CD8a ratios. We then examined the expression of CD39 and CD73 across diverse cell types, including myeloid cells, T lymphocytes, and B lymphocytes. Results: In both rat and mouse models, there were significant increases in the frequency of CD3+CD4+ T lymphocytes in PCAS (rat, P < 0.01; mouse, P < 0.001), with consequently elevated CD4/CD8a ratios in whole blood (both, P < 0.001). Moreover, CD39 and CD73 expression on blood leukocytes were markedly increased (rat, P < 0.05; mouse, P < 0.01 at 24h). Further analysis in the experimental mouse model revealed that CD11b+ myeloid cells, with significant increase in their population (P < 0.01), had high level of CD39 (88.80 ± 2.05 %) and increased expression of CD73 (P < 0.05). CD19+ B lymphocytes showed slight increases of CD39 (P < 0.05 at 2h) and CD73 (P < 0.05 at 2h), while, CD3+ T lymphocytes had decreased levels of them. These findings suggested a distinct patterns of expression of CD39 and CD73 in these specific immune cell populations after CA. Conclusions: These data have provided comprehensive insights into the immune response after CA, highlighting high-level expressions of CD39 and CD73 in myeloid cells.

Lupus autoantibodies initiate neuroinflammation sustained by continuous HMGB1:RAGE signaling and reversed by increased LAIR-1 expression.

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus, present in up to 80% of patients and leading to a diminished quality of life. In the present study, we used a model of lupus-like cognitive impairment that is initiated when antibodies that crossreact with excitatory neuronal receptors penetrate the hippocampus, causing immediate, self-limited, excitotoxic death of hippocampal neurons, which is then followed by a significant loss of dendritic complexity in surviving neurons. This injury creates a maladaptive equilibrium that is sustained in mice for at least 1 year. We identified a feedforward loop of microglial activation and microglia-dependent synapse elimination dependent on neuronal secretion of high mobility group box 1 protein (HMGB1) which binds the receptor for advanced glycation end products (RAGE) and leads to microglial secretion of C1q, upregulation of interleukin-10 with consequent downregulation of leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), an inhibitory receptor for C1q. Treatment with a centrally acting angiotensin-converting enzyme inhibitor or with an angiotensin-receptor blocker restored a healthy equilibrium, microglial quiescence and intact spatial memory.

Longitudinal patterns and predictors of response to standard-of-care therapy in lupus nephritis: data from the Accelerating Medicines Partnership Lupus Network.

BACKGROUND: Leveraging the Accelerating Medicines Partnership (AMP) Lupus Nephritis (LN) dataset, we evaluated longitudinal patterns, rates, and predictors of response to standard-of-care therapy in patients with lupus nephritis. METHODS: Patients from US academic medical centers with class III, IV, and/or V LN and a baseline urine protein/creatinine (UPCR) ratio ≥ 1.0 (n = 180) were eligible for this analysis. Complete response (CR) required the following: (1) UPCR < 0.5; (2) normal serum creatinine (≤ 1.3 mg/dL) or, if abnormal, ≤ 125% of baseline; and (3) prednisone ≤ 10 mg/day. Partial response (PR) required the following: (1) > 50% reduction in UPCR; (2) normal serum creatinine or, if abnormal, ≤ 125% of baseline; and (3) prednisone dose ≤ 15 mg/day. RESULTS: Response rates to the standard of care at week 52 were CR = 22.2%; PR = 21.7%; non-responder (NR) = 41.7%, and not determined (ND) = 14.4%. Only 8/180 (4.4%) patients had a week 12 CR sustained through week 52. Eighteen (10%) patients attained a week 12 PR or CR and sustained their responses through week 52 and 47 (26.1%) patients achieved sustained PR or CR at weeks 26 and 52. Week 52 CR or PR attainment was associated with baseline UPCR > 3 (ORadj = 3.71 [95%CI = 1.34-10.24]; p = 0.012), > 25% decrease in UPCR from baseline to week 12 (ORadj = 2.61 [95%CI = 1.07-6.41]; p = 0.036), lower chronicity index (ORadj = 1.33 per unit decrease [95%CI = 1.10-1.62]; p = 0.003), and positive anti-dsDNA antibody (ORadj = 2.61 [95%CI = 0.93-7.33]; p = 0.069). CONCLUSIONS: CR and PR rates at week 52 were consistent with the standard-of-care response rates observed in prospective registrational LN trials. Low sustained response rates underscore the need for more efficacious therapies and highlight how critically important it is to understand the molecular pathways associated with response and non-response.

Urine proteomic signatures of histological class, activity, chronicity, and treatment response in lupus nephritis.

Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%-40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

Peripheral T cell activation, not thymic selection, expands the T follicular helper repertoire in a lupus-prone murine model.

Many autoimmune diseases are characterized by the activation of autoreactive T cells. The T cell repertoire is established in the thymus; it remains uncertain whether the presence of disease-associated autoreactive T cells reflects abnormal T cell selection in the thymus or aberrant T cell activation in the periphery. Here, we describe T cell selection, activation, and T cell repertoire diversity in female mice deficient for B lymphocyte-induced maturation protein (BLIMP)-1 in dendritic cells (DCs) (Prdm1 CKO). These mice exhibit a lupus-like phenotype with an expanded population of T follicular helper (Tfh) cells having a more diverse T cell receptor (TCR) repertoire than wild-type mice and, in turn, develop a lupus-like pathology. To understand the origin of the aberrant Tfh population, we analyzed the TCR repertoire of thymocytes and naive CD4 T cells from Prdm1 CKO mice. We show that early development and selection of T cells in the thymus are not affected. Importantly, however, we observed increased TCR signal strength and increased proliferation of naive T cells cultured in vitro with antigen and BLIMP1-deficient DCs compared to control DCs. Moreover, there was increased diversity in the TCR repertoire in naive CD4+ T cells stimulated in vitro with BLIMP1-deficient DCs. Collectively, our data indicate that lowering the threshold for peripheral T cell activation without altering thymic selection and naive T cell TCR repertoire leads to an expanded repertoire of antigen-activated T cells and impairs peripheral T cell tolerance.

Host factor TIMP1 sustains long-lasting myeloid-biased hematopoiesis after severe infection.

Infection is able to promote innate immunity by enhancing a long-term myeloid output even after the inciting infectious agent has been cleared. However, the mechanisms underlying such a regulation are not fully understood. Using a mouse polymicrobial peritonitis (sepsis) model, we show that severe infection leads to increased, sustained myelopoiesis after the infection is resolved. In post-infection mice, the tissue inhibitor of metalloproteinases 1 (TIMP1) is constitutively upregulated. TIMP1 antagonizes the function of ADAM10, an essential cleavage enzyme for the activation of the Notch signaling pathway, which suppresses myelopoiesis. While TIMP1 is dispensable for myelopoiesis under the steady state, increased TIMP1 enhances myelopoiesis after infection. Thus, our data establish TIMP1 as a molecular reporter of past infection in the host, sustaining hyper myelopoiesis and serving as a potential therapeutic target for modulating HSPC cell fate.

Phenotypic and functional characteristics of murine CD11c+ B cells which is suppressed by metformin.

Since the description of age-associated or autoimmune-associated B cells (ABCs), there has been a growing interest in the role of these cells in autoimmunity. ABCs are differently defined depending on the research group and are heterogenous subsets. Here, we sought to characterize ABCs in Sle1/2/3 triple congenic (TC) mice, which is a well accepted mouse model of lupus. Compared to follicular (FO) B cells, ABCs have many distinct functional properties, including antigen presentation. They express key costimulatory molecules for T cell activation and a distinct profile of cytokines. Moreover, they exhibit an increased capacity for antigen uptake. ABCs were also compared with germinal center (GC) B cells, which are antigen activated B cell population. There are several phenotypic similarities between ABCs and GC B cells, but GC B cells do not produce proinflammatory cytokines or take up antigen. While T cell proliferation and activation is induced by both FO B and ABCs in an antigen-dependent manner, ABCs induce stronger T cell receptor signaling in naïve CD4+ T cells and preferentially induce differentiation of T follicular helper (Tfh) cells. We found that ABCs exhibit a distinct transcriptomic profile which is focused on metabolism, cytokine signaling and antigen uptake and processing. ABCs exhibit an increase in both glycolysis and oxidative phosphorylation compared to FO B cells. Treatment of ABCs with metformin suppresses antigen presentation by decreasing antigen uptake, resulting in decreased Tfh differentiation. Taken together, these findings define a fundamental connection between metabolism and function within ABCs.

The role of pyruvate-induced enhancement of oxygen metabolism in extracellular purinergic signaling in the post-cardiac arrest rat model.

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO2 production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood.

Lupus autoantibodies initiate a maladaptive equilibrium sustained by HMGB1:RAGE signaling and reversed by LAIR-1:C1q signaling.

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus (NPSLE), present in up to 80% of patients and leading to a diminished quality of life. We have developed a model of lupus-like cognitive impairment which is initiated when anti-DNA, anti-N-methyl D-aspartate receptor (NMDAR) cross- reactive antibodies, which are present in 30% of SLE patients, penetrate the hippocampus1. This leads to immediate, self-limited excitotoxic death of CA1 pyramidal neurons followed by a significant loss of dendritic arborization in the remaining CA1 neurons and impaired spatial memory. Both microglia and C1q are required for dendritic loss1. Here we show that this pattern of hippocampal injury creates a maladaptive equilibrium that is sustained for at least one year. It requires HMGB1 secretion by neurons to bind RAGE, a receptor for HMGB1 expressed on microglia, and leads to decreased expression of microglial LAIR-1, an inhibitory receptor for C1q. The angiotensin converting enzyme (ACE) inhibitor captopril, which can restore a healthy equilibrium, microglial quiescence, and intact spatial memory, leads to upregulation of LAIR-1. This paradigm highlights HMGB1:RAGE and C1q:LAIR-1 interactions as pivotal pathways in the microglial-neuronal interplay that defines a physiologic versus a maladaptive equilibrium.

B lymphocytes in treatment-naive paediatric patients with lupus are epigenetically distinct from healthy children.

BACKGROUND: SLE is likely triggered by gene-environment interactions. We have shown that most SLE-associated haplotypes encompass genomic regions enriched for epigenetic marks associated with enhancer function in lymphocytes, suggesting genetic risk is exerted through altered gene regulation. Data remain scarce on how epigenetic variance contributes to disease risk in paediatric SLE (pSLE). We aim to identify differences in epigenetically regulated chromatin architecture in treatment-naive patients with pSLE compared with healthy children. METHODS: Using the assay for transposase-accessible chromatin with sequencing (ATACseq), we surveyed open chromatin in 10 treatment-naive patients with pSLE, with at least moderate disease severity, and 5 healthy children. We investigated whether regions of open chromatin unique to patients with pSLE demonstrate enrichment for specific transcriptional regulators, using standard computational approaches to identify unique peaks and a false discovery rate of <0.05. Further analyses for histone modification enrichment and variant calling were performed using bioinformatics packages in R and Linux. RESULTS: We identified 30 139 differentially accessible regions (DAR) unique to pSLE B cells; 64.3% are more accessible in pSLE than healthy children. Many DAR are found in distal, intergenic regions and enriched for enhancer histone marks (p=0.027). B cells from adult patients with SLE contain more regions of inaccessible chromatin than those in pSLE. In pSLE B cells, 65.2% of the DAR are located within or near known SLE haplotypes. Further analysis revealed enrichment of transcription factor binding motifs within these DAR that may regulate genes involved in pro-inflammatory responses and cellular adhesion. CONCLUSIONS: We demonstrate an epigenetically distinct profile in pSLE B cells when compared with healthy children and adults with lupus, indicating that pSLE B cells are predisposed for disease onset/development. Increased chromatin accessibility in non-coding genomic regions controlling activation of inflammation suggest that transcriptional dysregulation by regulatory elements controlling B cell activation plays an important role in pSLE pathogenesis.

Not Dead Yet.

I have been a scientific grasshopper throughout my career, moving from question to question within the domain of lupus. This has proven to be immensely gratifying. Scientific exploration is endlessly fascinating, and succeeding in studies you care about with colleagues and trainees leads to strong and lasting bonds. Science isn't easy; being a woman in science presents challenges, but the drive to understand a disease remains strong.

Galantamine attenuates autoinflammation in a mouse model of familial mediterranean fever.

BACKGROUND: Autoinflammatory diseases, a diverse group of inherited conditions characterized by excessive innate immune activation, have limited therapeutic options. Neuroimmune circuits of the inflammatory reflex control innate immune overactivation and can be stimulated to treat disease using the acetylcholinesterase inhibitor galantamine. METHODS: We tested the efficacy of galantamine in a rodent model of the prototypical autoinflammatory disease familial Mediterranean fever (FMF). Multiple chronic disease markers were evaluated in animals that received long-term galantamine treatment compared to vehicle. RESULTS: Long-term treatment with galantamine attenuated the associated splenomegaly and anemia which are characteristic features of this disease. Further, treatment reduced inflammatory cell infiltration into affected organs and a subcutaneous air pouch. CONCLUSIONS: These findings suggest that galantamine attenuates chronic inflammation in this mouse model of FMF. Further research is warranted to explore the therapeutic potential of galantamine in FMF and other autoinflammatory diseases.

Thiocarbazate building blocks enable the construction of azapeptides for rapid development of therapeutic candidates.

Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics.